This invention relates generally to the treatment of diseases using nucleotide-based prodrugs. More particularly, the present invention relates to nucleotide-based prodrugs and their drug-delivery applications. The nucleotide-based prodrugs of the present invention comprise a drug component covalently attached via junctional ester bond(s) to one or more nucleotide components. Release and activation of the drug component of a nucleotide-based prodrug arises from hydrolysis of the junctional ester bond joining the nucleotide component to the drug component. The active drug component may be a nucleoside analog, a nucleic acid ligand, or a non-nucleoside drug. The nucleotide component provides a means of targeting and/or anchoring the nucleotide-based prodrug to the desired tissue compartment and/or a mechanism of sustained release of the active drug, thereby providing for a more effective drug delivery system with reduced toxicity. The targeting and/or anchoring of the nucleotide-based prodrugs to the desired tissue can be achieved through several methods, including employing a nucleic acid ligand as the nucleotide component, and/or by incorporating photocrosslinkable bases into the nucleotide component, and/or by covalently bonding the nucleotide component to a macromolecular support. The invention further includes lipid constructs comprising a nucleotide-based prodrug.
Many controlled release, tissue-targeted drug delivery systems have been developed and investigated in the laboratory, but few have reached the pharmaceutical marketplace. For reviews of this area of drug delivery research. (See Tomlinson, E. (1987) Adv. Drug Delivery Rev. 1:87-198 and Chein Y. W. 1992. Novel Drug Delivery Systems, New York, N.Y. Marcel Dekker, Inc. Many of the obstacles confronting scientists in this area of drug delivery research are illustrated by the examples provided below.
Insoluble Drug Depots
The most widely used slow release drug depots are contraceptive progestins implanted subcutaneously (e.g., Depo-Provera(copyright)). Some progestin formulations are encapsulated in permeable silicone chambers (e.g., Norplant(copyright)). Corticosteroid depots are also available (e.g., Depo-Medrol(copyright)). The deposition and sustained release of these drugs relies upon their relatively low solubilities in aqueous fluids. The steroids slowly dissolve at the surface of the implant, and diffuse into surrounding interstitium and capillaries. This sustained release technology is based entirely upon the drugs"" physicochemical properties, the geometry of the implant and its location in the tissue. The contraceptive""s target tissues (hypothalamus and adenohypophysis) are far removed from the depot, but low systemic drug levels are effective. The situation is different in the case of corticosteroid suspensions, which are used to treat locally inflamed tissues. A suspension of insoluble corticosteroid (DepoMedrol(copyright)) is injected directly into the tissue, so the targeting of the drug is crude. The drug""s absorption into local capillaries can lead to relatively high systemic levels and toxicity. Thus, only a few DepoMedrol(copyright) injections can be given to a patient each year.
Polymers Impregnated with Drugs
Drugs generally diffuse relatively rapidly from hydrated polymers. More avid drug sequestration is required to prolong drug release, but solving this engineering problem is very difficult. Each drug must be empirically matched to a polymer with a specific set of physicochemical properties. The polymer must not induce a macrophage-mediated foreign body reaction, and it must be non-immunogenic and chemically inert. Despite these constraints, a few products have been created (Dang, et al. (1996) Pharm. Res. 13:683-691). Gliadel(copyright) wafers (Guilford Pharmaceuticals) are used to deliver the alkylating agent bichloronitrosourea (BCNU) to brain tumors. After surgical resection of aggressive glioblastoma multiforme tumors, wafers are inserted into the cavity as a local adjuvant chemotherapy. Gliadel(copyright) wafers are comprised of a proprietary polymer impregnated with BCNU, which is released locally into peritumoral cerebral tissues for months after surgery. This method of dosing is crude, and unfortunately, Gliadel(copyright) doesn""t prolong survival more than a few months.
Hydrogels and other Polymers that Retard Drug Diffusion
Another strategy to localize drugs is to inject tissues with a polymer, such as a gel, soaked in a solution of the drug (Samuelov Y. et al. (1979) J. Pharm. Sci. 68:325-329; Graham, N. B. (1984) Biomaterials 5:27-36; Roorda, W. E. et al. (1986) Pharm Week [Sci] 8:165-189; Kaleta-Michaels, S. J. et al. (1994) Proc. Ann. Meet. Am. Assoc. Cancer Res. 35:A2473; Hnatyszyn, H. J. (1994) PDA J Pharm Sci Technol. 48:247-254; Nunes, G. L. et al. (1994) J. Am. Coll. Cardiol. 23:1578-83). Release of the drug into the surrounding tissue is limited by the viscosity of the gel, which isolates the drug from the interstitial fluid and local capillaries and thus retards diffusion. The rate of drug diffusion out of the gel is largely determined by the physical and biological properties of the gel, for example its hydrophobicity, tensile strength and biodegradability (Park, K. et al. (1993) Biodegradable Hydrogels for Drug Deliver. Technomic Publishing Co., Inc. Lancaster, Pa.). Several biological and non-biological gels are in various stages of development, including chimeric recombinant elastin-silk protein (Protein Polymers, Inc), collagen (Matrix Pharmaceuticals, Inc), poly-lactic acid (PLA), poly-glycolic acid, poly(xcex5-caprolactone), poly(xcex2-hydroxybutyrate), poly(xcex2-hydroxyvalerate), polydioxanone, poly(ethylene terephthalate), poly(malic acid), poly(tartronic acid), poly(ortho esters), polyanhydrides, polycyanoacrylates, poly(phosphoesters), polyphosphazenes, hyaluronidate, polysulfones, polyacrylamides, polymethacrylate, CarboPol and hydroxyapaptite (Park, K. et al. (1993) In most cases, the rate of drug release can be controlled by altering the gel""s water swelling capacity, tensile strength and rate of biodegradationxe2x80x94properties that can be adjusted by various chemical manipulations.
Biodegradable Hydrogels with Pendant Chains Covalently Coupled to Drugs
Controlled release from the above mentioned drug delivery systems is achieved by manipulating the physicochemical and biological properties of a polymer that is separate from the pharmacologically active drug molecule. Small drug molecules diffuse relatively rapidly through such hydrated polymers, thereby markedly limiting the duration of drug release. To address this shortcoming, investigators have sought to produce controlled release bioactive polymer systems comprised of biodegradable polymers covalently coupled to pharmacologically active drug molecules (reviewed in Bioactive Polymeric Systems (1985) Gebelein, G. C. and Carraher, C. E. Editors. Plenum Press, New York, N.Y.). Bioactive polymers have been synthesized by coupling pharmacologically active drugs to pendant chains via amide bonds or via more labile carbonyl ester linkages. Activation and release of the drugs requires hydrolysis of the amide or carbonyl ester bonds, and such hydrolytic reactions must occur at a rate that is slower than the rate of degradation of the biocompatible macromolecular support (i.e., the xe2x80x9cbackbonexe2x80x9d). Drugs have been attached to a variety of polymeric xe2x80x9cbackbonesxe2x80x9d, including starch microparticles (Laakso, T. et al. (1987) J. Pharm. Sci. 76: 134-140; Stjarnkvist, P. et al. (1991) J. Pharm. Sci. 80:436-440); poly(2-hydroxypropyl) methacrylamide copolymers (Duncan, R. et al. (1990) Biochem. Biophys. Res. Comm. 94: 284-290); poly-D-lysine (Shen, W-C et al. (1985) J. Biol. Chem. 260:10905-10908); and poly-N-(3-hydroxypropyl)-L-glutamine/leucine copolymers (Negishi, N. et al. (1987) Pharmaceutical Res. 4:305-310). Bioactive polymers wherein amide bonds link the drug to the pendant chain hydrolyze very slowly and they are relatively stable in serum and tissue fluids (Kopecik, J. (1984) Biomaterials, 5:19-25; Rejmanova et al. (1985) Biomaterials 6: 45-48). Typically, amide linked drugs are not efficiently released from the pendant chains or backbone unless the polymer is first engulfed by phagocytic cells, wherein the amide bonds are degraded in lysosomes. Drugs such as the narcotic naltrexone linked to poly-N-(3-hydroxypropyl)-L-glutamine/leucine copolymers via relatively labile carbonyl ester bonds are released at a nearly constant rate in vitro. The above mentioned examples are prodrugs whose activation requires hydrolysis of covalent bonds. While the amide and carbonyl ester bonds have succeeded in prolonging the duration of drug release, they have not provided a great deal of control over the rate of drug release, either because their rates of hydrolysis have been too stable (amide bonds) or too unpredictable (ester bonds).
Topical Drug Delivery
Topical drug therapy is used extensively for dermatological and ophthalmological applications (Wepierre, J. and Mary, J-P (1979) Trends in Pharm. Sci. 1:23-26; Guzzo, C. et al. (1996) Dermatological Pharmacology, In: Goodman and Gilman""s The Pharmacological Basis of Therapeutics, 9th Edition. Hardman, Limbird, Molinoff, Ruddon and Gilman, Eds., McGraw-Hill, USA pp.1593-1616.). Scores of topical drugs are available, including corticosteroids, retinoids, vitamin D3 analogs, anti-virals, anti-fungals, anti-bacterials, cytotoxic agents, antihistamines, analgesics and anesthetics. Most of the drugs are inexpensive and the applications are usually frequent. Topical delivery easily provides a constant rate of drug release for at least 24 hours (Transdermal Delivery of Drugs: Proceedings of the Workshop on Current Strategies and Future Directions. Washington D.C. U.S. Department of Health and Human Services. The National Institutes of Health. Higuchi, W. I. and Sharma D. editors).
Nevertheless, topical therapy has significant problems. Excessive application of topically administered retinoids, cholecalciferols and corticosteroids can lead to serious systemic side effects, because they diffuse rapidly into dermal capillaries. Drugs are absorbed more rapidly into the dermal capillaries of diseased skin, which may have reduced barrier function (Wepierre, J. supra). For example, psoriatic and eczematous skin lesions are much more permeable to topically applied drugs than normal skin.
Therapeutic levels of topically applied drugs may be difficult to achieve, because there is no mechanism to retain drugs in the skin after they have penetrated the stratum corneum, the principle barrier function of the skin. Drugs are absorbed into the dermal capillaries at a rate that far exceeds the rate of penetration through the stratum corneum. Consequently, it is exceedingly difficult to create a drug reservoir within the dermis and lower layers of the epidermis. Topical therapy on mucosal surfaces is ineffective, because some drugs are immediately absorbed into the submucosal capillaries. In addition, mucosal surfaces are easily desquamated and they are continuously cleansed with secretions. The lack of a sustained release mechanism limits the dose of many topical drugs, especially potent nonpolar agents. In addition, many drugsxe2x80x94especially macromolecular drugsxe2x80x94do not penetrate cornified layers of the epidermis, thereby precluding their use as topical agents. The topical route of administration cannot guarantee efficacious local drug concentrations.
Lipid-Based Drug Delivery
Lipid bilayer vesicles are closed, fluid-filled microscopic spheres which are formed principally from individual molecules having polar (hydrophilic) and non-polar (lipophilic) portions. (New, R. R. C. (1989) Preparation of Liposomes. In: Liposomes-a Practical Approach., IRL Press at Oxford University pp.33-104; Fielding, R. M. (1991) Clin Pharmacokinetics 21155-64; Gregoriadis, G. (1973) FEBS Lett. 36:292-296). The hydrophilic portions may comprise phosphato, glycerylphosphato, carboxy, sulfato, amino, hydroxy, choline or other polar groups. Examples of lipophilic groups are saturated or unsaturated hydrocarbons such as alkyl, alkenyl or other lipid groups. Sterols (e.g., cholesterol) and other pharmaceutically acceptable adjuvants (including anti-oxidants such as alpha-tocopherol) may also be included to improve vesicle stability or confer other desirable characteristics.
Liposomes are a subset of these bilayer vesicles and are comprised principally of phospholipid molecules that contain two hydrophobic tails consisting of fatty acid chains. Upon exposure to water, these molecules spontaneously align to form spherical, bilayer membranes with the lipophilic ends of the molecules in each layer associated in the center of the membrane and the opposing polar ends forming the respective inner and outer surface of the bilayer membrane(s). Thus, each side of the membrane presents a hydrophilic surface while the interior of the membrane comprises a lipophilic medium. These membranes may be arranged in a series of concentric, spherical membranes separated by thin strata of water, in a manner not dissimilar to the layers of an onion, around an internal aqueous space. These multilamellar vesicles (MLV) can be converted into Unilamellar Vesicles (UV) with the application of a shearing force.
The therapeutic use of liposomes includes the delivery of drugs which are normally toxic in the free form. In the liposomal form, the toxic drug is occluded, and may be directed away from the tissues sensitive to the drug and targeted to selected areas. Liposomes can also be used therapeutically to release drugs over a prolonged period of time, reducing the frequency of administration. In addition, liposomes can provide a method for forming aqueous dispersions of hydrophobic or amphiphilic drugs, which are normally unsuitable for intravenous delivery.
Amphiphilic and hydrophilic drug molecules are generally more efficiently encapsulated into liposomes than non-polar lipophilic drugs. Furthermore, certain non-polar lipophilic drugs and hydrophilic drugs tend to diffuse more rapidly out of liposomes in vivo compared to certain amphiphilic drugs, which may be firmly inserted into the lipid bilayer of the liposome.
In order for many drugs and imaging agents to have therapeutic or diagnostic potential, it is necessary for them to be delivered to the proper location in the body, and the liposome can thus be readily injected and form the basis for sustained release and drug delivery to specific cell types, or parts of the body. Several techniques can be employed to use liposomes to target encapsulated drugs to selected host tissues, and away from sensitive tissues. These techniques include manipulating the size of the liposomes, their net surface charge, and their route of administration. MLVs, primarily because they are relatively large, are usually rapidly taken up by the reticuloendothelial system (principally the liver and spleen). UVs, on the other hand, have been found to exhibit increased circulation times, decreased clearance rates and greater biodistribution relative to MLVs.
Passive delivery of liposomes involves the use of various routes of administration, e.g., intravenous, subcutaneous, intramuscular and topical. Each route produces differences in localization of the liposomes. Two common methods used in attempting to direct liposomes actively to selected target areas involve attachment of either antibodies or specific receptor ligands to the surface of the liposomes. Antibodies are known to have a high specificity for their corresponding antigen and have been attached to the surface of liposomes, but the results have been less than successful in many instances. Some efforts, however, have been successful in targeting liposomes to tumors without the use of antibodies (see, for example, U.S. Pat. No. 5,019,369). U.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled xe2x80x9cNucleic Acid Ligand Complexesxe2x80x9d (specifically incorporated herein by reference), describes nucleic acid ligands attached to the surface of liposomes which may be used as an alternative to antibodies for targeting purposes.
An area of development aggressively pursued by researchers is the delivery of agents not only to a specific cell type but into the cell""s cytoplasm and, further yet, into the nucleus (Ledley F. D. (1995) Human Gene Therapy 6:1129-44). This is particularly important for the delivery of biological agents such as DNA, RNA, ribozymes and certain proteins. A promising therapeutic pursuit in this area involves the use of antisense DNA and RNA oligonucleotides for the treatment of disease. However, one major problem encountered in the effective application of antisense technology is that oligonucleotides in their phosphodiester form can be quickly degraded in body fluids and by intracellular and extracellular enzymes, such as endonucleases and exonucleases, before the target cell is reached. Intravenous administration can also result in rapid clearance from the bloodstream by the kidney, and uptake is generally insufficient to produce an effective intracellular drug concentration. Liposome encapsulation protects the oligonucleotides from the degradative enzymes, increases the circulation half-life and increases uptake efficiency as a result of phagocytosis of the liposomes. In this way, oligonucleotides are able to reach their desired target and to be delivered to cells in vivo.
Antisense Oligonucleotides Conjugated to Lipids
Lipids have been used in other ways to improve the delivery of antisense nucleic acid oligomers. PCT Publication No. WO 90/10448 (Bischofberger) describes a conjugate comprising a lipid component covalently coupled to an antisense oligonucleotide. The lipid component provides a means for allowing the antisense oligonucleotide to cross cell membranes. Release of the antisense oligonucleotide from the lipid requires hydrolysis by cellular enzymes of the phosphodiester bonds (s) joining the lipid to the therapeutic oligonucleotide. Liposomes are also mentioned as a method for delivering the lipid-antisense oligonucleotides.
Lipid-Based Prodrugs Activated by Hydrolysis of Phosphodiester Bonds
A method of improving the intestinal absorption of drugs that are not orally bioavailable in a non-derivatized state is described in U.S. Pat. Nos. 5,411,947 and 5,484,809 (Hostetler). The oral delivery of these drugs is facilitated by converting drugs having suitable functional groups to 1-O-alkyl-, 1-O-acyl-, 1-S-acyl-and 1-S-alkyl-sn-glycero-3-phosphate derivatives. The active drug is released from the lipid prodrug by phospholipases that cleave the phosphoester bond linking the drug to the lipid.
U.S. Pat. No. 5,554,728 (Basava) describes peptide-lipid conjugates having increased plasma half-lives comprising therapeutic peptides covalently linked to lipids through linkers such as amino acids having hydroxyl functional groups.
U.S. Pat. No. 5,223,263 (Hostetler) describes lipid derivatives of nucleoside analogs which can be incorporated into liposomes. The lipid component allows the nucleoside analogs to be efficiently loaded and retained in liposomes and influences the biodistribution and pharmacokinetics of the nucleoside analogs. These derivatives are converted into active nucleoside analogs by constituent cellular metabolic processes.
U.S. Pat. No. 5,463,092 (Hostetler) describes phosphonoacidsxe2x80x94such as phosphonoformate (foscarnet)xe2x80x94having antiviral activity which are linked, either through a phosphate group or a carboxyl group of the phosphonic acid, to a lipid.
U.S. Pat. No. 5,614,503 (Chaudhary) describes an amphipathic nucleic acid transporter to deliver nucleic acids into cells, comprising a cationic compound having a cationic head group for binding the nucleic acid and a lipid tail for association with the cell membrane.
Simple Phosphoester-Based Prodrugs Activated by Phosphatases
The aqueous solubility of drugs may be increased by esterifying hydroxyl groups with negatively charged phosphates, which are removed by serum phosphatase(s), thereby activating the drug. These agents are herein referred to as simple phosphoester-based prodrugs, because favorable properties of such prodrugs are contributed by a phosphate. For example, the monophosphate derivative of the nucleoside fludarabine (9-xcex2-D-arabinofuranosyl-3-fluoro-adenine) (Chun H. G. et al. (1991) J. Clin. Oncol. 9:175-188) is more soluble in aqueous solutions than the non-phosphorylated fludarabine nucleoside. Intravenously administered fludarabine monophospate nucleotide is rapidly dephosphorylated in vivo by membrane 5xe2x80x2-ectonucleotidases; the resulting nucleoside is transported into the cell where it is rephosphorylated into the triphosphate derivative, which disrupts cellular nucleic acid metabolism.
Simple phosphoester prodrugs have also been created to improve the solubility properties and pharmacological properties of systemically administered non-nucleoside drugs. The oncolytic drug Etoposide(copyright) is a poorly soluble topoisomerase II inhibitor that must be formulated in polysorbate, polyethylene glycol and ethanol excipients (Doyle. T. W. and Vyas, D. M. (1990) Cancer Treat. Rev. 17:127-131; Schacter, L. P. et al. (1994) Cancer Chemother. Pharmacol. 34 (Suppl-S58-S63). To administer Etoposide(copyright) intravenously, the stock drug must be diluted to 0.2-0.4 mg/mL and infused over 30-60 minutes. This restriction is more than an inconvenience, because effective bolus IV doses cannot be used to treat cancer patients. To improve the aqueous solubility of Etoposide(copyright), the hydroxyl group on the phenyl ring is phosphorylated to create etoposide phosphate (Etopophos(copyright)). Five minutes after Etopophos(copyright) is intravenously administered, the phosphate is quantitatively removed by serum alkaline phosphatase, thereby activating the drug and allowing IV bolus dosing.
In other cases of phosphoester prodrugs, however, phosphoester hydrolysis is not nearly as efficient as in the above mentioned examples. For example, water soluble 2xe2x80x2- and 7xe2x80x2-taxol phosphates are poor substrates for alkaline phosphatase activity (Vyas et al., (1993) Bioorganic and Med. Chem. Lett. 3:1357-1360; Ueda, Y. et al. (1995) Bioorganic and Med. Chem. Lett. 5:247-252).
Covalent Cleavage (Release) of Drugs from Chemical Modifiers as a Method of Enhancing lontophoretic Drug Delivery
Prodrugs with improved properties for iontophoretic delivery have been described in U.S. Pat. No. 5,607,691 (Hale) which describes methods of delivering pharmaceutical agents across the skin layer or mucosal membranes of a patient. In this method, a pharmaceutical agent is covalently bonded to a chemical modifier, via a covalent physiologically cleavable bond. The chemical modifier comprises either permanently charged organic compounds or organic compounds which carry an ionic charge by virtue of the conditions of pH which exist transmembrane during transdermal delivery. The chemical modifiers function to alter the charge characteristics of a pharmaceutical agent in order to enhance membrane transport. The chemical modifier is cleaved from the pharmaceutical agent by a physiological process, thereby releasing the activated drug into the tissues.
The SELEX Process
A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed the SELEX process, is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichmentxe2x80x9d, now abandoned; U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled xe2x80x9cNucleic Acid Ligandsxe2x80x9d, now U.S. Pat. No. 5,475,096; U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled xe2x80x9cMethods for Identifying Nucleic Acid Ligandsxe2x80x9d, now U.S. Pat. No. 5,270,163 (see also WO 91/19813), each of which is specifically incorporated by reference herein. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for identifying nucleic acid ligands to virtually any desired target molecule. The SELEX process provides a class of products which are referred to as nucleic acid ligands (also referred to in the art as xe2x80x9captamersxe2x80x9d), each ligand having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled xe2x80x9cMethod for Selecting Nucleic Acids on the Basis of Structurexe2x80x9d, abandoned in favor of U.S. patent application Ser. No. 08/198,670, now U.S. Pat. No. 5,707,796, describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled xe2x80x9cPhotoselection of Nucleic Acid Ligandsxe2x80x9d, describes a SELEX based method, termed PhotoSELEX, for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking and/or photoinactiviating a target molecule. In one embodiment of the PhotoSELEX method, the photocrosslinkable ligand may be introduced into a patient in a number of ways known in the art. For example, the photocrosslinkable nucleic acid ligand may be delivered to the patient by intravenous injection of a solution or a lipid-based formulation (e.g., liposomes), by intralesional injection, by passive topical application, by iontophoresis or by other routes of administration. The ligand is then covalently crosslinked to the target molecules using irradiation, including visible, 325 nm, X-ray, ultraviolet and infrared light. Irradiation may be applied in vivo or ex vivo. High yield photocrosslinking occurs between the photoreactive bases juxtaposed to tyrosine, tryptophan, histidine and cystine residues within protein target molecules as demonstrated previously in many systems (Weintraub, H. (1973) Cold Spring Harbor Symp. Quant. Biol. 38:247; Lin and Riggs (1974) Proc. Natl. Acad. Sci. USA 71:947; Ogata and Gilbert (1977) Proc. Natl. Acad. Sci. USA 74:4973; Barbier et al. (1984) Biochemistry 23:2933; Wick and Matthews (1991) J. Biol. Chem. 266:6106; Khalili et al. (1988) EMBO J. 7:1205; Gott et al. (1991) Biochemistry 30:6290; Favre (1990) Bioorganic Photochemistry, Volume 1: Photochemistry and the Nucleic Acids. (H. Morrison, ed.) John Wiley and Sons., New York, pp.379-425; Evans et al. (1989) Biochemistry 28:713; Farrar et al. (1991) Biochemistry 30:3075; Wower et al. (1989) Biochemistry 28:1563; Liu and Verdine (1992) Tetrahedron Letters 33:4265; Chen and Prusoff.(1977) Biochemistry 16:3310).
U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled xe2x80x9cHigh-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,xe2x80x9d abandoned in favor of U.S. patent application Ser. No. 08/443,957 filed May 18, 1995, now U.S. Pat. No. 5,580,737, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, which can be non-peptidic, termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Solution SELEX,xe2x80x9d abandoned in favor of U.S. Pat. No. 5,567,588, describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled xe2x80x9cNucleic Acid Ligands to HIV-RT and HIV-1 Revxe2x80x9d, now U.S. Pat. No. 5,496,938, describes methods for obtaining improved nucleic acid ligands after the SELEX process has been performed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8, 1995, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEXxe2x80x9d, now U.S. Pat. No. 5,705,337, describes methods for covalently linking a ligand to its target. U.S. patent application Ser. No. 08/434,425, filed May 3, 1995, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Tissue SELEXxe2x80x9d, now U.S. Pat. No. 5,789,157 describes a method for identifying nucleic acid ligands to tissues.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands Containing Modified Nucleotidesxe2x80x9d, abandoned in favor of U.S. patent application Ser. No. 08/430,709 filed Apr. 27, 1995, now U.S. Pat. No. 5,660,985, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2xe2x80x2-positions of pyrimidines. U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2xe2x80x2-amino (2xe2x80x2-NH2), 2xe2x80x2-fluoro (2xe2x80x2-F), and/or 2xe2x80x2-O-methyl (2xe2x80x2-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled xe2x80x9cNovel Method of Preparation of Known and Novel 2xe2x80x2-Modified Nucleosides by Intramolecular Nucleophilic Displacementxe2x80x9d, describes oligonucleotides containing various 2xe2x80x2-modified pyrimidines. U.S. application Ser. No. 08/442,062, filed May 16, 1995, entitled xe2x80x9cMethods of Producing Nucleic Acid Ligandsxe2x80x9d, now U.S. Pat. No. 5,595,877, describes methods for identifying and designing improved nucleic acid ligands identified by the SELEX process.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEXxe2x80x9d, now U.S. Pat. No. 5,637,459, and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Blended SELEX,xe2x80x9d, now U.S. Pat. No. 5,683,867, respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic compounds of non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in U.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled xe2x80x9cNucleic Acid Ligand Complexesxe2x80x9d. Each of the above described patent applications, which describe the basic SELEX method and modifications of the basic SELEX method, are specifically incorporated by reference herein in their entirety.
Photochemotherapy to Trap Drugs in the Tissues
The most widely used form of photochemotherapy is psoralin-ultraviolet A (PUVA;) (Guzzo, C. et al., supra; Gonzalez, E. (1995) (1996) Dermatol Clin. 13:851-866). This method covalently fixes photoreactive psoralins to macromolecules within cells of the superficial dermis and epidermis. Psoralins easily penetrate all cells, and when activated by ultraviolet light (xcex=320 to 400 nm) they bind covalently to DNA and other molecules inside of cells. Psoralins eventually escape from cells that are not exposed to UVA, so this method has been used to xe2x80x98trapxe2x80x99 the drug specifically in the skin. Psoralins intercalate into DNA, and the majority of crosslinked products are psoralin-DNA adducts. Not surprisingly, psoralin adducts eventually overwhelm the nucleotide excision repair system that normally removes such lesions. Current evidence indicates that DNA crosslinking causes cell cycle arrest and/or apoptosis in proliferating cells, such as basal keratinocytes and lymphocytes, but more complex (cytokine mediated) effects are undoubtedly involved. DNA adducts have been found in keratinocytes, lymphocytes, dermal fibroblasts and Langerhans cells.
PUVA is approved for the treatment of cutaneous T-cell lymphoma (CTCL), psoriasis, lichen planus, atopic dermatitis, vitiligo, alopecia areata and cutaneous photosensitivity syndromes. In every case, the skin is characterized by abnormal lymphocytic infiltrates. CTCL is a neoplastic disease, whereas the others are immunoregulatory disorders. It remains unclear why these diseases respond to such a nonspecific therapy.
Many serious toxicities are associated with PUVA therapy (Wolff. K. (1990) Br. J. Dermatol. 122 Suppl 36:117-25; Young, A. R. (1990) J. Photochem. Photobiol. B. 6:237-47; Gonzalez, E. supra; Stern et al., (1997) New Eng. J. Med. 336:1041-1045). First, there is a ten-fold increase in squamous cell carcinoma and there is a significant increase in melanoma. Second, some patients suffer from burns, blisters, photoallergies and painful eythematous patches. Chronic treatment leads to mottled pigmentation and actinic skin damage. Finally, patients taking oral methoxsalen are at risk for developing cataracts, because methoxsalen penetrates the lens, where it may be crosslinked to crystalin proteins.
Photodynamic therapies (PDTs) have been developed to treat cancers of the bladder wall and a variety of malignant and benign skin conditions (Lui, H. et al. (1993) Dermatologic Clinics 11:1-13). PDTs utilize photoreactive porphyrin derivatives and dipyrroles, photoactivators that transfer their light energy to singlet oxygen, which damages cellular membranes and activates stress response pathways, some of which induce apoptosis. Unlike PUVA, the photoreactive drugs are not covalently crosslinked to cellular macromolecules.
Extracorporeal Photopheresis to Crosslink Drugs to Cells
An extracorporeal version of PUVA has been developed for the treatment of cutaneous T-Cell lymphoma (Edelson, R. et al. (1987) N. Engl. J. Med. 316:297-303). The therapeutic index of PUVA can be increased if the malignant T-cells are treated with UVA outside of the body. First, the patient is administered oral methoxsalen. Next, a semipure population of malignant cells is removed under sterile conditions by leukapheresis and are exposed to UVA. The cells are returned to the body, where many succumb to PUVA""s toxic DNA crosslinking effects, but it has been postulated that an active immunization phenomenon may also contribute to the therapeutic effects. However, the underlying mechanism of photopheresis remains poorly understood. Photopheresis is most effective in CTCL patients who have a significant leukemic component (e.g., Sezary Cells).
Clearly, it would be useful to have more effective methods of concentrating drugs in diseased tissue compartments. Systemic toxicities of many potent drugs would be minimized or eliminated by providing sustained release of the drug from a tissue reservoir located within or immediately adjacent to the diseased tissue compartment, and by markedly reducing the volume of drug distribution. It would also be useful to have a local delivery method whereby active drugs are gradually released from tissue reservoirs and which do not displace normal tissue structures. For example, a new technology that provides for the gradual release of drugs at the dermo-epidermal junction would bypass the cornified layers at the surface of the skin, which comprises the major rate limiting barrier to diffusion of topically administered drugs. Furthermore, tissue-anchoring and sustained release of drugs into a small volume within the sub-epithelial dermis would minimize the rapid diffusion and absorption problems that limit the usefulness of current topical drug delivery methods, such as passive diffusion and iontophoresis. Finally, a powerful and versatile sustained drug release technology would utilize known chemistry for the release of the active drug and for the manipulation of the rate of the chemical reaction controlling drug release and activation.
The use of nucleic acid polymers as a component of a prodrug in which the nucleic acid polymer simultaneously provides a means of targeting and/or anchoring the prodrug to the target and provides a mechanism for sustained release of the active drug component has not been taught in the prior art. The present invention demonstrates that nucleotide-based prodrugs possess numerous advantages over current drug delivery systems.
The present invention provides novel nucleotide-based prodrugs comprising a nucleotide component covalently bonded to a drug component via a junctional ester bond. In one embodiment, the junctional ester bond is a physiological hydrolyzable ester bond. The present invention exploits the ability to control the rate of hydrolysis of this junctional ester bond as a means of providing a controlled sustained release of the active form of the drug.
The nucleotide component of the nucleotide-based prodrugs of the present invention provides the novel and versatile properties of targeting and/or anchoring the nucleotide-based prodrug in the immediate vicinity of the target (i.e., the desired site for therapy) and providing for sustained release of the pharmacologiclly active form of the drug component. Release of the active drug arises from hydrolysis of a junctional ester bond which joins the active drug component to the nucleotide component of the nucleotide-based prodrug. The present invention is based on the unique insight that the rate of release and activation of the drug can be manipulated by chemically altering the nuclease resistance of the nucleotide component of the nucleotide-based prodrug and/or chemically altering the nuclease resistance of the junctional ester bond joining the drug to a nucleotide component. Increasing the nuclease resistance of the nucleotide component will in turn decrease the rate of release of the active drug component of the nucleotide-based prodrug. In this way, versatile sustained release properties can be bestowed upon nucleotide-based prodrugs.
The ability to manipulate of the rate of hydrolysis of ester bonds in the nucleotide component is significant in that it distinguishes the nucleotide-based prodrugs of the present invention from other classes of prodrugs. Unmodified oligonucleotides are rapidly degraded in vivo by tissue nucleases and esterases. However, methods are known for modifying nucleotides to make them more resistant to degradation in vivo. The inventors have exploited these methods to develop novel nucleotide-based prodrugs, wherein the release and activation of a drug component can be prolonged by strategically modifying the nucleotide component of the nucleotide-based prodrug. A variety of chemical modifications which will increase the nuclease resistance of nucleotide components are known in the art. In one embodiment of the invention, nucleotide residues in the nucleotide component are modified at the 2xe2x80x2-position of the sugar moiety in order to make the nucleotide component more resistant to nucleases, phosphodiesterases and other esterase activities in vivo. In other embodiments of the present invention, the rate of hydrolysis of the nucleotide component can be markedly prolonged by modifying the phosphodiester backbone of the nucleotide component. Such modifications will confer stability upon the junctional ester bond which joins the active drug component to the nucleotide component of the nucleotide-based prodrug.
The anchoring and sustained release properties of the nucleotide-based prodrugs of the present invention have the additional benefits of allowing for a high, local concentration of the drug at or near a desired target. Thus, after administration of the nucleotide-based prodrug the nucleotide component is gradually hydrolyzed by nucleases and/or other phosphodiesterases in the tissue and the active drug component is released in the vicinity of the target. This establishes a desirable drug gradient (see, for example, FIG. 3). Furthermore, since the nucleotide-based prodrug is anchored at or near the desired target, the diffusion distance of the active drug component to the target is minimized once the drug has been liberated from its prodrug form. As a result, the active drug is delivered directly to the desired target, resulting in reduced systemic toxicity. Therefore, the nucleotide-based prodrug of the present invention allows exposure of the target to a high, local concentration of a drug over an extended period of time, thus reducing both the size and frequency of doses of the drug necessary for effective treatment, providing for a wide therapeutic index and providing for a more effective therapy. Thus, the nucleotide-based prodrugs of the present invention overcome the inherent shortcomings of current drug delivery systemsxe2x80x94such as rapid diffusion, rapid absorption of drugs and systemic toxicityxe2x80x94by providing a unique method of targeting and/or anchoring the nucleotide-based prodrug at or in the vicinity of the target.
In certain embodiments of the present invention, local delivery of the nucleotide-based prodrugs and sustained release of active drug components are achieved without excipients, resins or gels, so there is virtually no tissue displacement upon administration of the nucleotide-based prodrug.
In certain embodiments, the nucleotide component of the nucleotide-based prodrugs comprises a nucleic acid ligand (aptamer) which performs a dual function of targeting and anchoring the nucleotide-based prodrug to a desired target by virtue of aptamer-specific binding to the target. Examples of this embodiment are illustrated in FIGS. 1B-H. In a preferred embodiment, the nucleic acid ligand is identified by the SELEX process, and the nucleic acid ligand binds the nucleotide-based prodrug to macromolecules such as proteins in a diseased tissue. Thus the nucleotide-based prodrugs in this embodiment are distinguished from other prodrugs in that the present nucleotide-based prodrugs precisely target the therapy.
In another embodiment, the nucleotide-based prodrug is anchored at or in the vicinity of the desired target by incorporating photoreactive nucleotides into the nucleotide component. In this embodiment, the nucleotide-based prodrug is administered locally, and the site of administration is exposed to UV light, which induces photocrosslinking between a target, for example a protein in a tissue, and the photoreactive nucleotides. Examples of this embodiment are illustrated in FIGS. 2D-F.
In other embodiments, nucleotide-based prodrugs are sequestered in discrete tissue sites by virtue of the covalent attachment of the nucleotide component to a poorly diffusible macromolecular support (FIGS. 2A-C). In the preferred embodiment, the macromolecular support is a biocompatible polymer.
In the above embodiments, it is necessary that the junctional ester bond coupling the drug component to the nucleotide component of the nucleotide-based prodrug is able to be hydrolyzed in vivo.
In another embodiment, the nucleotide-based prodrug comprises radioactive nucleotides as the drug component. In this embodiment, it may be desirable to prevent junctional ester bond hydrolysis and therefore prevent release of the radioactive nucleotides in order to avoid toxicity of certain tissues.
The invention further includes nucleotide-based prodrugs with amphiphilic properties. These nucleotide-based prodrugs comprise pharmacologically active, non-polar lipids, such as retinoids, vitamin D analogs, eicosanoids, steroids or ceramide analogs covalently bonded to one or more nucleotide components via junctional ester bonds (FIG. 24). Hydrolysis of the junctional ester bonds in vivo releases either the active lipid compound or a monophosphorylated derivative of the active lipid compound that is subsequently activated by tissue phosphatases. In one embodiment, the amphiliphilic nucleotide-based prodrug is administered in a lipid vehicle comprised of a lipid bilayer vesicle, preferably a liposome.
In embodiments wherein the pharmacologically active prodrug component of the nucleotide-based prodrug is covalently bonded to more than one nucleotide component, the release of the drug will be prolonged even further since release of the drug will require the hydrolysis of more than one junctional ester bond.
The present invention also includes methods for treating diseases in humans and animals by administering pharmaceutically effective amounts of a nucleotide-based prodrug. Nucleic acid prodrugs are administered by methods known in the art. In embodiments wherein the nucleotide-based prodrug contains a nucleic acid ligand component or wherein the nucleotide-based prodrug is in a lipid bilayer vesicle, the nucleotide-based prodrug may be administered systemically or locally. In embodiments wherein the nucleotide-based prodrug comprises a non-aptameric nucleotide component comprising one or more photoreactive basesxe2x80x94or where the non-aptameric nucleotide component is covalently bonded to a poorly diffusible macromolecular supportxe2x80x94the nucleotide-based prodrug is administered, for example, at the site of the diseased tissue, and the site of application is exposed to UV light to induce covalent bond formation between the photoreactive bases with the desired target.